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Adenoviral Vectors for Gene Therapy of Inherited and Acquired Disorders of the Lung
Published in Kenneth L. Brigham, Gene Therapy for Diseases of the Lung, 2020
David T. Curiel, Robert I. Garver
The details of plasmid design and common methods of use have been described in recent reviews (18,19) and therefore will not be reiterated in detail here (Fig. 1). In brief, the most widely used method involves three major steps. First, the new coding sequence with appropriate transcriptional start and stop regulatory sequences is added to a multiple cloning site within the deleted El region of a plasmid containing a portion of the left-hand (5') end of the adenovirus genome. Second, this plasmid vector containing the new coding sequence is cotransfected into 293 cells with a second plasmid that contains the entire adenovirus genome with an El deletion modified to contain a “stuffer fragment” of plasmid DNA. The stuffer fragment not only contains the plasmid origin of replication and antibiotic resistance gene for bacterial propagation, but it is sufficiently large to prevent that adenoviral DNA from being packaged into a stable viral particle. Homologous recombination occurs be tween the two plasmids so that the El region containing the coding sequence of interest replaces the plasmid stuffer within the otherwise intact genome, and the E1 proteins made by the 293 cells activate the recombinant genome replication with the result that recombinant virus is made. The third step is a series of plaque purifications with screening assays at each step to eliminate undesired wild type virus that is generated by homologous recombination between the viral sequences within 293 cells and the adenoviral plasmid with the stuffer fragment.
Use of Enzymes in the Downstream Processing of Biopharmaceuticals
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Precise enzyme-catalyzed structural modifications have also targeted key impurities with the goal of facilitating their separation from the target molecule. Two notable examples can be found in the case of the very challenging purification of minicircle (MC) DNA vectors. MCs are produced in vivo in Escherichia coli by first replicating a parental plasmid (PP) backbone and then promoting an intramolecular site-specific recombination between two strategically placed multimer resolution sites (Simcikova et al., 2016). This generates a replication-deficient MC, which contains the expression cassette with therapeutically useful transgene, and a miniplasmid (MP), which contains the prokaryotic replication segment used for PP amplification. The separation of MCs from the MP impurities is very difficult to perform since the two species are very similar in terms of structure and size (Alves et al., 2016).
Vector Technology of Relevance to Nitrogen Fixation Research*
Published in Peter M. Gresshoff, Molecular Biology of Symbiotic Nitrogen Fixation, 2018
Reinhard Simon, Ursula B. Priefer
Consequently, pLAFR1 or pLAFR3 (with an additional multiple cloning site168a) has been the cosmid vector of choice in the construction of gene banks of various Rhizobium strains, including R. leguminosarum,169,170R. meliloti 41,171Brady rhizobium japonicum,172,173R. phaseoli,174 and Azorhizobium sesbania ORS571.175
Surface-modified vacuole-based daunorubicin delivery system for acute myeloid leukaemia (AML) and their selective therapeutics
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2022
Wooil Choi, Yang-Hoon Kim, Jiho Min
The backbone vector, pYES2::SS::MCS::VMA11::His-tag, for the protein expression on the outer membrane of yeast vacuole was constructed. Briefly, the original vector pYES2 (Invitrogen, USA) is used as a platform to build the target vector that can express any protein of interest on the yeast vacuolar outer membrane. Different parts of polypeptide VMA11, including signal sequence fragment (SS) (consisting of the first 39 amino acids), Part 2 fragment (P2) (consisting of the last 46 amino acids) and Part 1 fragment (P1) (consisting of the next 79 amino acids), are in turn inserted into pYES2. The sequence of P1 and P2 has been reversed from the original sequence. The multi cloning site (MCS) is located between SS and P2. The TLR2 binding peptide (T2BP) and enhanced green fluorescence protein (GFP) were inserted in this site.
Cancer cells under immune attack acquire CD47-mediated adaptive immune resistance independent of the myeloid CD47-SIRPα axis
Published in OncoImmunology, 2021
Mark A.J.M. Hendriks, Isabel Britsch, Xiurong Ke, Anne P. van Wijngarden, Douwe F. Samplonius, Emily M. Ploeg, Wijnand Helfrich
A detailed evaluation of bsAb CD47xEGFR-IgG, including its construction and production, was recently published by our group.8 In short, DNA fragments encoding scFv MABL and scFv 425 were generated by commercial gene synthesis service (GenScript) based on published VH and VL sequence data.20,2122 For construction and production of bsAb CD47xEGFR-IgG2s, we used eukaryotic expression plasmid pEE14-bsAb, which contains three multiple cloning sites (MCS) for directional and in-frame insertion of DNA fragments: scFv MABL, scFv 425 and IgG2s-Fc.11 BsAb CD47xEGFR-IgG2s was produced using the Expi293 expression system (ThermoFisher). Expi293 supernatant, containing bsAb CD47xEGFR-IgG2s, was purified using a HiTrap protein A HP column connected to an ÄKTA Start chromatography system (GE Healthcare Life Sciences).
Bispecific antibody approach for EGFR-directed blockade of the CD47-SIRPα “don’t eat me” immune checkpoint promotes neutrophil-mediated trogoptosis and enhances antigen cross-presentation
Published in OncoImmunology, 2020
Mark A. J. M. Hendriks, Emily M. Ploeg, Iris Koopmans, Isabel Britsch, Xiurong Ke, Douwe F. Samplonius, Wijnand Helfrich
DNA fragments encoding scFv MABL and scFv 425 were generated by commercial gene synthesis service (Genscript) based on published VH and VL sequence data.18,19 For construction and production of bsAb CD47xEGFR-IgG, we used eukaryotic expression plasmid pEE14-bsAb, which contains 3 consecutive multiple cloning sites (MCS). MCS#1 and MCS#2 are interspersed by a 22 amino acid flexible linker.20 MCS#1, MCS#2 were used for directional and in-frame insertion of DNA fragments encoding scFv MABL, scFv 425, and MCS#3 for insertion of DNA fragments encoding human Fc IgG1, IgG2s21 or IgG4, respectively (Figure S1a,b). Analogously, pEE14-CD47xMock-IgG1 encoding bsAb CD47xMock-IgG1 was constructed by replacing scFv425 in pEE14-CD47xEGFR-IgG1 by scFv4-4-20 directed against fluorescein.22